Our long-term goal is to elucidate th mechanism(s) by which the hormone melatonin and its receptor(s) transmit signals regarding environmental light/dark changes to the central nervous system, with particular emphasis on the role of melatonin in modulating circadian rhythms in mammals. This proposal focuses on the localization, regulation, and structural properties of melatonin receptors. In these studies we will use the chicken brain, which is highly enriched in ML-1 melatonin receptors which are primarily localized in visual areas of the chicken brain. We will determine by quantitative autoradiography with the selective radioligand 2-[125I]- iodomelatonin the density of 2-[125I]-iodomelatonin binding sites in the various layers of the optic tectum as well as in areas of the chicken brain receiving retinal projections following optic nerve transection. These experiments together with studies of melatonin receptor axonal transport following ligation of the optic nerve will test the hypothesis that a population of 2-[125I]-iodomelatonin binding sites are localized on presynaptic axon terminals. Build-up of receptors at the site of ligature will allow pharmacological analysis by autoradiography of transported receptors destined for axon terminals. We will also determine the diurnal patterns of melatonin binding sensitivity (affinity and density), and the role of endogenous melatonin in regulating these receptor sites in selected areas of the chicken brain. The ability of receptors to act presynaptically to modulate the release of dopamine, norepinephrine, and serotonin in slices from chicken brain areas containing high levels of these monoamines will be investigated using well established superfusion protocols. The pharmacology of the presynaptic melatonin receptor sites will be determined using selective melatonin receptor agonists, antagonists (e.g., luzindole) and partial agonists (e.g., 50CH3-luzindole). We will purify and determine the molecular properties of the high affinity melatonin receptor of chicken brain. In order to accomplish this goal we will synthesize melatonin- and luzindole-related analogues bearing carboxyl or amino groups that will allow for preparation of affinity columns and probes for photoaffinity labeling that will permit identification and pharmacological characterization of the purified receptor. In summary, the studies proposed herein should provide us with novel and important insights concerning the pharmacological, functional and biochemical properties of melatonin receptors. Results from this proposal will advance our understanding of melatonin receptor(s) function and may provide the foundation to clone the melatonin receptor. This may lead to the discovery of novel therapeutic treatments for various physical and mental illnesses that are associated with disorders of the circadian timekeeping.